Due to the amphiphilic properties of medium chain aliphatic alcohols, they have very important applications in the industry, which can be used in the fields of surfactants, medicines, cosmetics and energy sources, with a market value of 100-120 million US dollars. The aliphatic hydrocarbon molecules having a carbon chain length of 6-16 are the main components of aviation kerosene, with advantages of high calorific value, low vapor pressure, low freezing point, low hygroscopicity, etc. 50% of commercial fatty alcohols are extracted from plant seeds or animal fats, and the remaining fatty alcohols and all hydrocarbons are refined from petroleum. Neither method is capable of meeting the requirements of sustainable and environment-friendly production by the modern society. On the contrary, with the rapid development of synthetic biology, genetically engineered bacterial strains can specifically synthesize the required products using renewable energy resources sugar, xylan, glycerol or the like.
In engineered E. coli, aliphatic alcohols and hydrocarbons are mainly derivatively synthesized from the fatty acid synthesis pathway. Respectively, three molecules, aliphatic acyl-ACP/CoA and free fatty acid, can be used as synthetic precursors. Conversion of aliphatic acyl-ACP/CoA or fatty acids to aliphatic aldehydes in the synthesis of hydrocarbon alcohols is a critical step, followed by reduction of aliphatic aldehydes to aliphatic alcohols or by decarbonylation reactions to become hydrocarbons with one carbon less. Microbial synthesis of aliphatic alcohols/hydrocarbons using aliphatic acyl-ACP/CoA as precursors has been reported since 2010. However, the artificial synthesis system for synthesizing medium chain hydrocarbon alcohols using free fatty acids as substrate only appeared in two reports until 2013. Howard et al. over-expressed thioesterase from Cinnamomum camphora in E. coli, released free fatty acids of specific length from aliphatic acyl-ACP, and simultaneously expressed the fatty acid reductase (FAR) encoded by the genes of luxC, luxD, luxE from Photorhabdus luminescens and fatty aldehyde decarbonylase from Nostoc punctiforme PCC73102, thereby the free fatty acids were reduced to aliphatic aldehydes and subsequentially decarboxylated into hydrocarbon molecules with one carbon less, and a hydrocarbon synthesis system using free fatty acid as the substrate was constructed, which was able to synthesize a relatively controllable length. Akhtar et al. discovered that the carboxylic acid reductase (CAR) from Mycobacterium marinum was able to convert the free fatty acids having a chain length ranging from C6 to C18 to the corresponding aliphatic aldehydes. This enzyme can be combined with an aliphatic aldehyde reductase or an aliphatic aldehyde decarboxylase to produce an aliphatic alcohol having an even numbered chain length (C8-C16) and a hydrocarbon compound having an odd numbered chain length (C7-C15) in vitro. The E. coli BL21 (DE3) strain is able to synthesize up to 350 mg/L of fatty alcohols with glucose as the carbon source in the minimum medium when such pathway is combined with a thioesterase capable of producing free fatty acid of a specific chain length in the cell.
Since the above two types of hydrocarbon synthesis systems using free fatty acids as the substrate both employed reductase for aldehyde reaction, they are called the reduction type hydrocarbon synthesis systems. Because under the same substrate conditions, the reductase requires the reducing power (NAD(P)H) and energy (ATP) provided by cells to perform reaction, while the reaction driving force of oxidase is provided by the oxygen molecules, oxidative synthesis system is a more economical microbial synthesis system. Currently, there has been no related work yet published on the artificially synthetic construction of oxidation-type hydrocarbons.
On the other hand, in the currently reported works, hydrocarbon alcohol artificial synthesis systems, whether taking aliphatic acyl-ACP/CoA or free fatty acid as the precursor, as the first step of the reduction reaction does not involve decarbonylation reaction, all the synthesized aliphatic alcohols are of even numbered carbon chains, while all the hydrocarbon molecules are of odd numbered carbon chains as a result of the one-step decarbonylation reaction. In fact, all the petroleum-based chemicals and fuels have diversity in structure, and simultaneously contain molecules of straight and branched chains, as well as odd and even numbered chains. An ideal biofuel should be both structurally and chemically similar to existing petroleum-based fuels. There has been work to alter the upstream fatty acid synthesis pathway for downstream synthesis of branched chain and even numbered chain alkanes. But there has been no work for directly regulating the downstream synthesis pathway.